Dfc450 c digital microscope camera

Liver samples were preserved in neutral buffered formalin (10 %) before use. Formalin-fixed tissues were embedded in paraffin, sectioned (5 μm), and stained with hematoxylin and eosin (H&E). H&E stained liver sections were evaluated by light microscopy at 200 X magnification for evidence of hepatocyte apoptosis, inflammation, and fibrosis using a Leica DMI3000 B Inverted Light Microscope System with Leica DFC450 C Digital Microscope Camera (Leica Microsystems, Inc.).

Cell migration was assayed with 8-μm-pore size Transwell migration chambers (Thermo Fisher Scientific) (Bronkhorst et al., 2014 (link)). Monocytes (10⁶ cells) in 1 ml of DMEM/Ham’s F-12 with antibiotics and 10% human serum or in 1 ml of CM were added to the upper chamber. In the lower chamber, the chemokine CCL2 (100 ng/ml, Peprotech EC Ltd, London, United Kingdom) was added to the medium. Heparan sulfate proteoglycan could influence the effects of CM on cell migration (Shute, 2012 (link)). Therefore, we included experimental groups treated with CM previously incubated with anti-heparan sulfate proteoglycan antibody (Sigma-Aldrich, clone A7L6, 10 μg/ml) for 2 h. Cell migration was allowed to proceed for 72 h at 37°C and 5% CO₂. Then, inserts were separated and migrated cells were washed with PBS, observed in a microscope Leica DM IL LED (Leica Microsystems, Solms, Germany), and photographed with a Leica DFC450 C Digital Microscope Camera using Leica Application Suite software. Cells were quantified using the Cell counter complement of ImageJ (NIH, United States).